SENP3‐mediated TIP60 deSUMOylation is required for DNA‐PKcs activity and DNA damage repair

Abstract The activation of DNA‐dependent kinase (DNA‐PKcs) upon DNA damage contains a cascade of reactions, covering acetylation by TIP60, binding with Ku70/80, and autophosphorylation. However, how cells regulate TIP60‐mediated acetylation of DNA‐PKcs and the following DNA‐PKcs activation upon DNA damage remains obscure. This present study reported that TIP60 is hyper‐SUMOylated in normal conditions, but upon irradiation‐induced DNA damage, small ubiquitin‐like modifier (SUMO)‐specific protease 3 (SENP3)‐mediated deSUMOylation of TIP60 promoted its interaction with DNA‐PKcs to form the TIP60‐DNA‐PKcs complex. We show that TIP60 SUMOylation is reduced quickly in response to DNA damage and the deSUMOylation of TIP60 by SENP3 is required for DNA‐PKcs acetylation and its autophosphorylation. Comet and γH2AX immunofluorescence assay showed that knockdown of SENP3 impaired DNA damage repair. Using the NHEJ report system, we found that knockdown of SENP3 affected the efficiency of NHEJ. Further exploration using clonogenic survival assay, cell viability assay and cytoflow assay suggested that leaking SENP3 increased the sensitivity of tumour cells to serval DNA damage treatment. Overall, our findings revealed a previously unidentified role of SENP3 in regulating DNA‐PKcs activity and DNA damage repair.

which is active throughout the cell cycle. 2,3 However, the HR pathway, different from NHEJ, is viewed as an errorfree repair pathway that requires an intact homologous template and occurs mainly in the S/G2 phase. 4,5 In addition, the DSBs activate a complex network of cellular pathways named the DNA damage response (DDR) pathways, including the sense, signalling, repairing of DNA lesions, cell cycle checkpoints and apoptosis. [6][7][8][9] DDR is a signal transduction pathway, which involves diverse forms of post-translational protein modifications, including acetylation, methylation, SUMOylation, phosphorylation and ubiquitination. 10 The function of modification via SUMO (small Ubiquitin-like MOdifier) is crucial to sustaining genomic integrity, modulating transcriptions, expressing genes, and mediating signal transduction between cells. 11,12 Similar to ubiquitination, SUMOylation has been discovered in multiple DDR (DNA damage response)-associated proteins, such as BRCA1, BLM, 53BP1 and Rad52. 10,13,14 However, unlike deubiquitination, the function of deSUMOylation in DDR is far from illusion. In mammalian cells, there are 7 SUMO proteases (SENP1-7) that can process SUMO conjugates. 15 DSB is reported to dissociate SENP6 from RPA70, so that SUMO2/3 can modify the RPA70, which prompts the Rad51 recruitment to result in HR initiation. 16 SENP1 deSUMOylates the SIRT1 K734 site, inhibiting the deacetylase activity of SIRT1 in cells exposed to UV radiation. 17 Also, SENP3 has been reported to deSUMOylate NPM1, promoting BRCA1 recruitment and HR repair in response to DNA damage. 18,19 DNA-PKcs refers to a serine/threonine-protein kinase which belong to the phosphatidylinositol-3-kinase like kinase (PIKK) family and becomes a master regulator of the NHEJ pathway. 20 Once DSBs occur, the Ku heterodimer (Ku70 and Ku80) rapidly senses it and binds to the broken ends in a sequence-independent manner. 21,22 Next, the catalytic subunit of DNA-PKcs gets quickly recruited to the DSBs sites and is activated by the Ku heterodimer followed by the activation of a set of NHEJ downstream components, containing Artemis, XRCC4, and DNA ligase IV (Lig4), localised to the DSBs sites. 23 The activity of DNA-PKcs is tightly regulated by its posttranslation modifications. Upon DNA damage, DNA-PKcs can be phosphorylated at over 40 sites. Among them, the T2609 and S2056 clusters are best characterised. 24,25 In addition to the phosphorylation modification, other forms of post-translational modifications are also present on DNA-PKcs, such as PARylation, 26 Neddylation 27 and TIP60-dependent DNA-PKcs acetylation.
Initially recognised as a protein of 60 kDa, TIP60, also referred to as KAT5, is a member of the MYST family of HATs (histone acetyltransferases), which is linked to the HIV Tat. 28 TIP60 participates in multiple cellular pro-cesses, such as chromatin reconstruction, gene transcription, apoptosis, maintain genomic stability 29 and DNA repair. 30 Once DNA double strands break occurs, TIP60 localises itself on the damaged sites and acetylates H3, H4 and γH2AX, leading to chromatin relaxation and remodelling. [31][32][33][34] TIP60 also interacts with ATM and DNA-PKcs through its acetylase activity in response to DNA damage, eventually stimulating their kinase activity. 32,35 We recently revealed that PISA4 E3 ligase SUMOylates TIP60 at the K430 site, which can be conjugated by SENP3. Thus, blocking its interaction with DNA-PKcs leads to the inhibition of DNA-PKcs kinase activity, in turn, promoting the HR repair pathway in the S phase cells. 35 The current work aimed to explain the role of SENP3mediated TIP60 K430 deSUMOylation in the regulation process of DNA-PKcs activity and DNA repair. Using the Co-Immunoprecipitation (Co-IP) assay, we identified that upon irradiation-induced DNA damage, a decrease was observed in the TIP60 K430 SUMOylation, which was conjugated with SENP3. This facilitated the binding of TIP60 and DNA-PKcs. Importantly, SENP3 knockdown impaired the DNA-PKcs activity and NHEJ repair. Upon knocking down SENP3, cancer cells became strongly sensitive to DNA damaging drugs and irradiation. Altogether, we provided new insights into the molecular mechanisms that controlled the DNA-PKcs activity upon DNA damage. We also identified a possible target for the treatment of tumour.

The interaction between DNA-PKcs and TIP60 was increased upon irradiation-induced DNA damage
As a critical constituent of DNA damage signalling, TIP60 is involved in the elicitation of both ATM and DNA-PKcs upon DNA damage. The MRN complex has been reported to recruit both ATM and TIP60 to DNA damage sites, facilitating the formation of the TIP60-ATM complex. The DNA-PKcs localisation to DSBs sites is dependent on the Ku dimer, which also competes with MRN for DNA ends to determine which repair pathway is used. This indicates that the mechanism underlying the regulation of TIP60 and DNA-PKcs binding upon DNA damage is likely to be distinct from ATM.
To investigate the mechanism underlying the modulation of interplay between TIP60 and DNA-PKcs in the event of DNA damage, we initially utilised the extracts of cells (chromatin-free) from the HeLA cells transfected with Flag-TIP60 plasmid to conduct the Co-IP assay using the anti-FLAG antibodies. Interestingly, the F I G U R E 1 The interaction between DNA-PKcs and TIP60 is increased upon irradiation-induced DNA damage. (A) After transitory transfection using Flag-tagged TIP60, the HeLA cells were subjected to treatment either with or without 4 Gy γ-ray irradiation, 1 h later, anti-Flag affinity gel was used to accomplish immunoprecipitation of the harvested cell lysates, and designated DNA-PKcs antibody was used to conduct western blot. (B) The TIP60-DNA-PKc interplay in non-radiated and 1 h radiated (4 Gy) HeLA cells was validated through co-immunoprecipitation (Co-IP) assays using either anti-Flag or DNA-PKcs antibody. The Co-IP samples were subjected to SDS-PAGE isolation and subsequent immunoblotting for the designated proteins. (C) E. coli (BL21) bacterial expression of TIP60 based on the GST-pull-down assay. The pull-down productions were western blotted using the DNA-PKcs antibody. (D) E. coli (BL21) bacterial expression of DNA-PKcs H domain (AA3540-4128) based on the GST-pull-down assay. The western blotting of pull-down productions was performed with the TIP60 antibody. (E) After transitory transfection using designated plasmids, the HEK-293T cells were subjected to treatment under 4 Gy γ-ray irradiation, followed by collection and separate 0.5-and 1-h lysing treatments. Flag beads were utilised to pull down the SUMOylated TIP60 proteins, which were then examined through Western blot TIP60-DNA-PKcs interplay increased dramatically after the irradiation ( Figure 1A). Simultaneously, further Co-IP assay using anti-DNA-PKc antibodies also revealed enhancement of DNA-PKc-TIP60 interplay after irradiation ( Figure 1B).
Next, we intended to confirm these results using the GST pull-down assay. To our surprise, when we used the TIP60 protein expressed in B121 E. coli to perform the GST pulldown test of DNA-PKcs, no DNA damage-dependent alteration was observed during the interaction of TIP60 and DNA-PKcs ( Figure 1C). Then, we performed the GST pulldown assay again using the C-terminal AA3540-4128 (H domain) of DNA-PKcs, which was the interaction domain of DNA-PKcs with TIP60 expressed in Bl21 E. coli. Here, we found that the DNA-PKcs interaction increased dramatically with the TIP60 protein in the extract of HeLA cells after irradiation ( Figure 1D). It is suggested that the increase in the interaction between TIP60 and DNA-PKcs in response to the DNA damage could not be reliant on DNA-PKcs but on the post-translation modification of TIP60 that could not occur in the B121 E. coli.
Considering that the TIP60 K430 SUMO2 modification attenuated its interaction with DNA-PKcs in S phase cells, this study wondered whether this modification was also responsible for the regulation of TIP60 and DNA-PKcs interaction in response to the irradiation. To verify our prediction, a Co-IP assay was conducted to identify the SUMOylation of TIP60 after irradiation, those result showed that the SUMOylation of TIP60 was lowered dramatically after IR ( Figure 1E).

SNEP3 knockdown attenuated the interaction between TIP60 and DNA-PKcs upon irradiation
Previously, 35 we observed that SENP3 could mediate de-SUMOylation of the TIP60 K430 site. To demonstrate whether SENP3 affected the TIP60 de-SUMOylation after irradiation, we performed Co-IP assays in this study. As shown in Figure 2A, SENP3 knockdown abolished the decrease of TIP60 SUMOylation after IR. In order to rule out the off-target impacts of the SENP3 siRNA, this study reconstituted the SENP3 siRNA-treated cells with a siRNAresistant wild-type (WT) SENP3 expression plasmid. As confirmed by the obtained results, the siRNA-resistant SENP3 rescued the TIP60 SUMOylation, which was earlier conferred by the siRNA (Figure 2A).
Given SENP3 deSUMOylates TIP60 upon irradiationinduced DNA damage, we proposed the hypothesis that SENP3 promotes TIP60-DNA-PKcs interaction upon irradiation. To identify this, we performed Co-IP assays, whose results showed that knocking down of SENP3 with siRNA dramatically attenuated TIP60-DNA-PKcs interaction after treating the cells with 4 Gy irradiation, which could be rescued by expressing siRNA-resistant wild-type (WT) SENP3 ( Figure 2B). Since TIP60-DNA-PKcs interaction was essential for TIP60-mediated DNA-PKcs acetylation upon DNA damage, the DNA-PKcs acetylation level was also detected in the SENP3 knockdown cells. Similar to the binding of TIP60 and DNA-PKcs, knocking down of SENP3 with siRNA dramatically weakened the irradiation-induced DNA-PKcs, but not ATM and Histone H4, acetylation, which could be rescued by expressing siRNA-resistant wild-type (WT) SENP3 ( Figure 2C).
Since the TIP60 K430 SUMOylation plays a vital role in its binding with DNA-PKcs, we explored whether the SUMOylation modification at the TIP60 K430 site regulated its interaction with DNA-PKcs and activated DNA-PKcs upon irradiation. Hence, we performed the Co-IP assays, as shown in Figure 2D. The TIP60 K430R mutation showed a stronger binding affinity to DNA-PKcs but not to ATM, which was activated independently of DNA damage in TIP60 K430R mutant cells. In comparison with TIP60 WT, TIP60 K430R mutant cells showed higher level of DNA-PKcs pS2056, which is a symbol for DNA-PKcs activity, and γH2AX, but not ATM pS1981, in untreated HeLA cells ( Figure 2E). This indicates TIP60 K430R mutant could activate DNA-PKcs independent of DNA damage. Next, we also detected the phosphorylation of DNA-PKcs substrate CHK2 and found that CHK2 T68 phosphoryla-tion also increased without irradiation in the TIP60 K430R mutant cells ( Figure 2F). Based on the obtained results, TIP60 K430 SUMOylation not only regulates DNA-PKcs activity in a cell cycle-dependent pathway but also exerts a vital role in DNA-PKcs activity upon DNA damage.

2.3
Knockdown of SENP3 decreased the DNA-PKcs activity upon DNA damage For the purpose of investigating the effects of SENP3 on the activation of DNA-PKcs, this study carried out an immunofluorescence assay to detect the localisation of TIP60, DNA-PKcs, and DNA-PKcs pS2056 on DNA damage sites. We noted that the TIP60 K430R mutant did not influence the recruitment of both TIP60 and DNA-PKcs to DNA damage sites ( Figure 3A-D). However, the DNA-PKcs pS2056 was dramatically decreased by knocking down SENP3 ( Figure 3E and F). Western blotting results showed that ATM pS1981 was enhanced in both SENP3 WT and SENP3 knockdown cells in response to DNA damage. Nevertheless, the radiation-induced pS2056 in DNA-PKcs was significantly attenuated in SENP3 siRNA-treated cells, which could be recovered by expressing si-Res SENP3 ( Figure 3G). Thus, our results showed that knocking down of SENP3 had a moderate effect on DNA-PKcs pT2609, which indicated that the T2609 cluster was primarily targeted by Ataxia telangiectasia mutated (ATM) or ATM and Rad3 related (ATR) mechanisms but not DNA-PKcs itself.

Knockdown of SENP3 inhibited NHEJ efficiency and DNA repair
On the basis of the preliminary results, it could be observed that the knockdown of SENP3 decreased the auto-phosphorylation of DNA-PKcs after irradiation. From this, it can be assumed that the activation of downstream proteins of DNA-PKcs is also reduced. To investigate this hypothesis, we performed the Immunofluorescence assay with 4 Gy irradiation, where the cells were harvested after irradiating for 1 h. Our results showed that the foci of Artemis could be impaired by the DNA-PKcs inhibitor or could be significantly reduced by the knockdown of SENP3 ( Figure 4A and B). Our results further proved that knocking down of SENP3 inhibited the activation of DNA-PKcs, affecting the downstream protein activation.
Given the vital roles of DNA-PKcs and Artemis in DSBs repair via the NHEJ pathway, we reckon that knocking down SENP3 may abate NHEJ efficiency. We performed both the NHEJ and HR assays. Upon knocking down of SENP3 with siRNA, the results showed dramatically reduced efficiency of the NHEJ pathway, whereas no F I G U R E 2 Knocking down of SNEP3 attenuates the interaction between TIP60 and DNA-PKcs upon irradiation. (A) Knocking down SENP3 using two single siRNA against SENP3 (SENP3 siRNA1 and siRNA2) or Control siRNA in HEK-293T cells (transfected either using both HA-SUMO2 and Flag-TIP60 plasmids or not), and re-expressed SENP3 using SENP3 siRes. This was followed by treatment of cells in the presence or absence of irradiation with 4 Gy γ-ray. The cells were then harvested and lysed at 1 h post-irradiation, and the next step was Co-IP assay with Flag beads to detect SUMOylated TIP60 proteins. SDS-PAGE was conducted for sample isolation, and sample incubation was accomplished using designated antibodies. (B) Knocking down SENP3 using two single siRNA against SENP3 (SENP3 siRNA1 and siRNA2) or Control siRNA in HEK-293T cells (transfected either using Flag-TIP60 plasmids or not), and re-expressed SENP3 using SENP3 siRes. Subsequently, cell treatment proceeded in the presence or absence of 4 Gy γ-ray irradiation. The cells were then harvested and lysed at 1 h after irradiation, followed by assessment of TIP60-DNA-PKc interaction via the Co-IP assay utilising Flag beads. (C) Knocking down SENP3 using two single siRNA against SENP3 (SENP3 siRNA1 and siRNA2) or Control siRNA in HEK-293T cells, and re-expressed SENP3 using SENP3 siRes. This was followed by treatment of cells in the presence or absence of irradiation with 4 Gy γ-ray. The cells were then harvested and lysed at 1 h after irradiation. Next step was the Co-IP assay using DNA-PKcs or ATM antibodies. Western blot proceeded subsequently using designated antibodies, ATM and H4K16Ac as controls. (D) After transitory transfection using designated plasmids (Flag-TIP60 WT or Flag-TIP60 K430R), the HEK-293T cells were subjected to treatment in the presence or absence of irradiation with 4 Gy γ-ray. The cells were then harvested and lysed at 1 h post-irradiation. Later, the TIP60-DNA-PKc interplay was examined through Co-IP assay using Flag beads. (E) After transitory transfection using K430R or Flag-TIP60 WT mutants, the HeLA cells were subjected to treatment in the presence or absence of irradiation with 4 Gy γ-ray, followed by collection and separate 0.5-and 1-h lysing treatments. SDS-PAGE was conducted for sample isolation, while sample incubation was accomplished using designated antibodies. (F) After transitory transfection using K430R or Flag-TIP60 WT mutants, the HeLA cells were treated with or without 4 Gy γ-ray irradiation, followed by collection and a 1-h lysing treatment. SDS-PAGE was conducted for sample isolation, while sample incubation was accomplished using designated antibodies F I G U R E 3 Knocking down of SENP3 decreases the DNA-PKcs activity upon DNA damage. (A) Twenty-four hours following transfection using SENP3 siRNA or control, the HeLA cells were irradiated with or without 4Gy γ-ray. The TIP60 and γH2AX expressions were examined 1 h later, which was accomplished through immunofluorescence assay using corresponding antibodies. (B) Quantification of the foci numbers of γH2AX and TIP60 in the HeLA cells post 4 Gy γ-ray irradiation. Data were represented as means ± SDs of triplicate (at least) experiments. In every experiment, scoring was made on 50 cells. ns means no significance. (C) Twenty-four hours following transfection using SENP3 siRNA or control, the HeLA cells were subjected to treatment with or without 4 Gy γ-ray irradiation. The DNA-PKcs and γH2AX expressions were examined 1 h later, which was accomplished through immunofluorescence assay with corresponding antibodies. (D) Quantification of the foci numbers of γH2AX and DNA-PKcs in the HeLA cells post 4 Gy γ-ray irradiation. Data were represented as means ± SDs of triplicate (at least) experiments. In every experiment, scoring was made on 50 cells. ns means no significance. (E) Twenty-four hours following transfection using SENP3 siRNA or control, the HeLA cells were subjected to treatment with or without 4 Gy γ-ray irradiation. One hour later, immunofluorescence assay was performed to test the expression of DNA-PKcs pS2056 and γH2AX using corresponding antibodies. (F) Quantification of the foci numbers of γH2AX and DNA-PKcs pS2056 in the HeLA cells post 4 Gy γ-ray irradiation. Data were represented as means ± SDs of triplicate (at least) experiments. In every experiment, scoring was made on 50 cells. ns means no significance,**p < 0.01. (G) Knocking down SENP3 using two single siRNA against SENP3 (SENP3 siRNA1 and siRNA2) or Control siRNA in HeLA cells, and re-expressed SENP3 using SENP3 siRes. Subsequently, treatment of cells proceeded in the presence or absence of irradiation with 4 Gy γ-ray. The resulting cells were then harvested and lysed at 1 h after irradiation. SDS-PAGE was conducted for sample isolation, while sample incubation was accomplished using designated antibodies F I G U R E 4 Knocking down of SENP3 inhibits NHEJ efficiency. (A) Twenty-four hours following transfection using SENP3 siRNA or control, the HeLA cells were treated with or without 4 Gy γ-ray irradiation. One hour later, immunofluorescence assay was performed to test the expression of Artermis and γH2AX using corresponding antibodies. (B) Quantification of the foci numbers of γH2AX and Artemis in the HeLA cells post 4 Gy γ-ray irradiation 1 h (50 cells each point in each experiment). Data represented are means ± SDs of triplicate (at least) experiments. ns means no significance. **p < 0.01. (C) DR-GFP (direct repeat GFP) reporter assay was conducted to examine the HR efficiency, as expounded in the Methods part. Data represented are means ± SDs of triplicate (at least) experiments. ns means no significance. **p < 0.01. (D) EJ5-GFP reporter assay was employed to assess the NHEJ efficiency. The positive control was BRCA1 siRNAs, whereas the negative control was 53BP1 siRNAs. Data represented are means ± SDs of triplicate (at least) experiments. ns indicates insignificance. *p < 0.05, **p < 0.01. (E, F) Neutral comet assay of SENP3 siRNA-or control-transfected HeLA cells, 24 h later, cells at NO IR, 0, 4, and 8 h post 4 Gy γ-ray irradiation treatment. The histogram displays average quantitation of tail moment from 50 nuclei picked randomly. The error bars indicate SDs. ns means no significance. **p < 0.01. (G) Twenty-four hours following transfection using SENP3 siRNA or control, the HeLA cells were subjected to irradiation with 4 Gy γ-ray, followed by collection at the designated times. Immunofluorescence assay was performed to test the expression of γH2AX using corresponding antibodies. (H) Quantification of the foci numbers of γH2AX in HeLA cells at various points of time following irradiation with 4 Gy γ-ray (50 cells each point in each experiment). Data are means ± SD from at least triplicate experiments, ns indicates insignificance. *p < 0.05; **p < 0.01 influence was observed on the efficiency of the HR pathway ( Figure 4C and D). Then, we also performed the comet ( Figure 4E and F) and γH2AX immunofluorescent assays ( Figure 4G and H) to measure the function of SENP3 in DNA repair. Overall, the DNA repair efficiency was significantly reduced in SENP3 knockdown cells.

2.5
Knockdown of SENP3 could exacerbate the outcome of cancer therapy by irradiation and DNA damaging drugs In order to deeply investigate the role of SENP3 in DNA damage, we performed the cell flow assay to detect the effect of SENP3 in HeLA cell apoptosis after irradiation. As shown in Figure 5A and B, a dramatic increase was observed in the apoptosis percentage of cancer cells with SENP3 knockdown after being treated with irradiation. Next, the results of the cell viability and cell colony formation assays showed that knocking down SENP3 could reduce cell viability in different cells using different DNA damage drugs such as Cisplatin, Camptothecin (CPT), Etopophos (ETO) and Mitomycin C (MMC). Especially, upon inducing DNA damage by the TOP1 inhibitor CPT, mainly repaired by the NHEJ pathway, the cell viability was increased to the level of the control group when backfilled with SENP3 ( Figure 5C and D). In the cell colony assay, we found that both HeLA and MD231 cells showed the same results. An increased radiation dose showed that the cell survival rate in the SENP3 knockdown cells was much lower than that of the SENP3 WT cells. However, backfilling the cells with siRNA-resistant SENP3 brought back the cell survival rate to the normal level ( Figure 5E and F).

SENP3 was upregulated in cancer and associated with worse prognosis
Many biological processes require reversible posttranslational protein modification through incorporation of SUMO proteins. SUMO precursors are first processed by SENP3 and other SUMO-specific proteases, so that the C-terminal diglycine motif necessary for the splicing event can be produced. These proteases hold isopeptidase activity as well, enabling SUMO elimination from the molecularly heavy SUMO couples. 36 We examined whether the levels of SENP3 are linked to the carcinoma development among patients. In fact, in the aforementioned 4 datasets, the expression of SENP3 are obviously higher in tumour tissues than in adjacent non-tumour tissues ( Figure 6A-D), implying that higher expression of SENP3 were related to tumourigenesis. Kaplan-Meier analysis demonstrated that higher SENP3 levels in tumour tissues were obviously correlated with the enhanced overall survival (OS) rates in BLCA, CHOL, LIHC and SARC cancers ( Figure 6E-H).

DISCUSSION
TIP60-DNA-PKcs interaction is essential for the NHEJ repair 37 but the mechanism of how DNA damage regulates TIP60-mediated DNA-PKcs activity is far from revealed.
Here, we demonstrated that TIP60 is hyper-SUMOylated in normal conditions, but upon irradiation-induced DNA damage, SENP3 deSUMOylates TIP60, which promotes its interaction with DNA-PKcs and the latter activity. SENP3 is significant for the efficiency of NHEJ repair. Knocking down of SENP3 impairs DNA repair efficiency, and such tumour cells become hypersensitive to radiotherapy and chemotherapy.
Overall, we demonstrated that TIP60 K430 SUMO2 modification mediated its interaction with DNA-PKcs and used the NHEJ repair mechanism for irradiationinduced DNA damage in a cell cycle-dependent manner 35 ( Figure 7). As an acetylase, TIP60 exerts a vital function in the DNA damage response through acetylating multiple substrates, including DNA-PKcs, ATM, P53, Histone H4 etc. 32,38,39 However, the mechanism of how DNA damage activates TIP60 is still unclear. Also, we demonstrated that TIP60-DNA-PKcs complex formation is promoted by irradiation in a SENP3-dependent manner. SENP3 deSUMOylates TIP60 K430 SUMO2 modification which inhibits its binding to DNA-PKcs. Our study revealed new mechanisms, which indicated that DNA damage regulates downstream of TIP60 by mediating their interaction. However, whether the ability of other substrates to interact with TIP60 is regulated by DNA damage or other conditions need further investigation.
According to literature reports, the post-translational SUMO protein modifications are responsible for modulating diverse protein functions, such as chromatin configuration, protein stability, transcription, subcellular localisation, DNA repair, inter-protein interactions as well as proteostasis. 12,[40][41][42][43][44] The SUMO precursor processing and uncoupling are modulated by SENP (Sentrin/SUMOspecific protease), thereby achieving the cellular machinery control. 18 However, unlike DUB(Deubiquitination), the role of SENPs in DNA damage repair is far from being revealed.
SENP3 is a SENP family member, which enables protein modification alteration by uncoupling the target proteins. 45 To keep SUMOylation balanced, the modification by SUMO and SENP3 should be effective, which also helps guarantee normal cellular activities and protein functionality. 46 In the physiological context, the role of SENP3 is a molecule responding to oxidative stress. Contrastively, in the pathological context, SENP3 may elicit a cellular response in case the fluctuations in its level influence the protein SUMOylation process, which results in cellular activity anomalies, as well as the disease contraction and development (e.g. neurological conditions, cardiovascular conditions, and various carcinomas). 18 Nevertheless, the role of SENP3 in DNA damage repair still remains unclear. The SENP3 has been reported to deSUMOylate NPM1, which promotes HR repair. This implies that the role of SENP3 is inhibiting the HR pathway. 47 We also identified SENP3 as a positive regulator of DSBs repair, which functions in the NHEJ pathway by promoting TIP60-DNA-PKcs interaction and DNA-PKcs activity upon DNA damage.
As an essential kinase in NHEJ repair, DNA-PKcs activity is mediated by several post-translational modifications, containing phosphorylation, Neddylation, PARyla-tion, acetylation etc. [24][25][26][27] Among these, TIP60-mediated DNA-PKcs acetylation is essential for DNA-PKcs activity upon DNA damage, and we have reported that TIP60-DNA-PKcs interaction is regulated in a cell-cycledependent manner, which is attenuated in the S phase. Even though it has been pointed out that TIP60-DNA-PKcs interaction is not enhanced by Bleomycin which induces DNA damage by inhibiting DNA synthesis in S phase cells, our data showed that TIP60-DNA-PKcs interaction was increased upon irradiation-induced DNA damage. This indicated that the regulation of TIP60-DNA-PKcs interaction might be used as a guideline for drugs chosen for tumour therapy. 48 Our results showed that the SENP3 knockdown tumour cells were most sensitive to CPT, a TOP1 inhibitor that induced DNA damage in and around the cell cycle but not as a DNA synthesis inhibitor.
Briefly, this work reveals that SENP3-mediated TIP60 K430 deSUMOylation regulates its interaction with DNA-PKcs and also influences the latter's activity, playing a vital

Antibodies and chemicals
The

RNA interference target sequences
Synthesis of siRNAs was accomplished by the GenePharma Biotech in Shanghai. Regarding the transfection procedure, the indicated siRNA was used to accomplish twice transfection of cells with Lipofectamine 2000 (Invitrogen) at a 24-h interval as per the protocol of manufacturer. The siRNA sequences are listed as follows: SENP3-siRNA1: GGCGUGUCAGUUGAUGAAAdTdT; SENP3-siRNA2: CUGGAAAGGUUACAAAdTdT; 53BP1-siRNA: GAGAGCAGAUGAUCCUUUAdTdT; BRCA1-siRNA: CAGCUACCCUUCCAUCAUAUUdTdT.

Western blotting
Following collection and lysis of cells, a 10-min heating of the protein samples at 100 • C was accomplished in fivefold loading buffer. Next, the cell lysates were subjected to SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) isolation and subsequent shift onto nitrocellulose membranes. This was followed by blockage of membranes with skimmed milk (5%) in onefold TBST, and a 1-h (or overnight) incubation at ambient temperature (or 4 • C) using primary antibodies. Thereafter, the membranes were thrice washed in onefold TBST and then incubated for 1 h using secondary antibodies at ambient temperature. Finally, the membranes were washed again in 1× TBST three times.

Co-Immunoprecipitation
As a first step of Co-IP assay, cell lysis was accomplished using the NETN buffer [300 mM NaCl, 20 mM Tris-base, 1 mM EDTA and 0.5% (v/v) NP-40] involving onefold protease inhibitor cocktail (cOmplete). After a 20-min constant agitation at 4 • C, the cell lysates were subjected to a 10-min centrifugation at 12,000 rpm, followed by harvesting and a 5-h incubation of the supernatant using corresponding antibodies or beads with rotation under a 4 • C condition. Thereafter, thrice washing of the samples proceeded in NETN buffer. The final immunoprecipitants were denatured in a 2× loading buffer for 15 min at 100 • C and analysed by immunoblotting.

HR and NHEJ DNA repair assay
The efficiencies HR and NHEJ DNA repair were examined via HR and NHEJ assays as per the reported protocols. Initially, the indicated siRNA was transfected into U2OS cells that incorporated DR-GFP (or EJ5-GFP) reporters, which were presents from Teng Ma's lab. Then, p-cherryand I-Scel-expressing vectors were transfected into the cells. This was followed by incorporation of DOX for the I-SecI expression elicitation, and 48 h later, FACS was employed to examine the proportions of GFP-or RFPpositive cells. The efficiencies of HR and NHEJ repair were represented by GFP-positive cells as percentages of RFP-positive cells, whereas the frequencies of repair were expressed as means ± SDs of triplicate experiments (at least).

Immunofluorescence assay
Following coverslip cultivation in dishes (35 mm), treatment of cells was accomplished using ionising radiation (IR) at a dose of 4 Gy. Then, the cells were subjected to PBS (phosphate-buffered saline) rinsing, paraformaldehyde (3%) fixation at ambient temperature for 12-15 min, and a 30-min permeabilisation using Triton X-100 (0.3%) in onefold PBS at ambient temperature. The next step was blockage of antibody binding sites (non-specific) using BSA (3%) in onefold PBS, and a subsequent 60-min incubation of cells at ambient temperature using antibodies. After thrice washing in onefold PBS, a further 60-min incubation of cells proceeded under dark and ambient temperature conditions using secondary antibodies. Thereafter, the slides were subjected to thrice washing in onefold PBS and a 10-min DAPI staining at ambient temperature for the nuclear DNA visualisation. Finally, placement of coverslips was accomplished on anti-fade buffer-containing slides, and a fluorescence microscope (Nikon) was utilised for result visualisation.

Clonogenic survival assay
Six hours following seeding in dishes (60 mm), the HeLA and MD231 cells were subjected to SENP3 siRNA transfection and irradiation at predesigned doses. For formation of colonies, incubation of the cells was accomplished in medium (4 ml), as well as 2 weeks of cultivation. After crystal violet (0.5%) staining of cells in 20% methanolinvolving PBS, the colonies containing over 50 cells were quantified.

Comet assay
Cells were seeded and maintained for 48 h and then collected and resuspended in phosphate-buffered saline (PBS) at a concentration of 3 × 10 5 cells per ml. The comet assay was then performed according to the manufacturer's instructions. DNA damage was measured using Cometscore software in terms of the length of the tail movement.

GST-pull down assay
During the GST pull-down assays of TIP60 or DNA-PKcs-H, purification of fusion protein fragments was accomplished on glutathione Sepharose 4B beads following expression in the E. coli (BL21) cells. The next step was a 6-h incubation of the purified proteins at 4 • C using cell lysates. After SDS-PAGE resolution of the resulting proteins, they were subjected to Western blot analysis.

Apoptosis assay
As for the cell apoptosis, the instructions of manufacturer in the Annexin V, FITC Apoptosis Detection Kit (DOjindo) were followed. Briefly, cells were resuspended in 500 μl binding buffer according to the instructions and incubated with 5 μl Annexin V-FITC and 5 μl PI in a dark environment and the number of apoptotic cells was recorded using a flow cytometer from FACSCalibur (ACEA Biosciences).

Cell viability assay
Each 2000 HeLA and MD231 cells were seeded per well of 96-well microplates and transfected with SENP3 siRNA or SENP3 siRNA + SENP3 siRES, then treated with cisplatin, etoposide, campathecin or mitomycin C. Viability assay was accomplished 2 days later with the CellTiter-Blue reagent (Promega) for these cells. Data expressed were means ± SDs of triplicate experiments (at least).

Statistical analysis
The mRNA-seq data were constructed by exploiting the dataset from TCGA tumours (Portal https://tcga-data.nci. nih.gov/tcga/). Spearman's tests were conducted to examine both the tumour and normal tissue levels of SENP3 among patients. Survival plots were estimated through Kaplan-Meier technique, while the log-rank test was employed for determination of significance at a * p < 0.05. The results were computed by exploiting quantitative data from triplicate experiments, which are represented as means ± SDs. SPSS v18.0 was utilised to statistically analyse results via one-way analysis of variance. LSD t-test was employed to assess the significance of the inter-group differences. The differences with * p < 0.05 and ** p < 0.01 were considered statistically significant.

A C K N O W L E D G E M E N T
We acknowledge Teng Ma provided the DR-GFP and EJ5-GFP reporters.

C O N F L I C T O F I N T E R E S T
All authors declare no conflict of interests.

A U T H O R C O N T R I B U T I O N S
S.G., H.Y., X.H.,X.C. performed most of the experiments and analysed the data. Y.C., L.G., J.J., M.S., G.L., D.X., H.G., S.Z., X.L., H.Z. assisted with the critical regents and provided technical help. S.G., P.Z., H.G. conceived the project and wrote the manuscript.

D ATA AVA I L A B I L I T Y S TAT E M E N T
All data are available from the corresponding authors upon request.